Constant cell renewal in epithelial tissues is accomplished by a carefully balanced process in which new cells are constantly being produced in exact measure to the number of cells that are lost through cell death. Following wounding, a burst of mitotic activity takes place so that the number of cells produced outnumbers the cells that are dying. As the tissue mass is being replenished, mitotic activity slows so that eventually a new equilibrium between cell production and cell loss is obtained. Although it is clear that such process requires both positive and negative growth factors, the molecules involved in controlling epidermal homeostasis have not yet been characterized (Choi, Y. and Fuchs, E., Cell Regulation, 1:791-809 (1990)). Transforming growth factors of the .beta. type (TGF.beta.)s are candidates to be major regulators in this process since it has been shown that both TGF-.beta.1 and TGF-.beta.2 can inhibit epidermal proliferation at ng/ml concentrations (Choi, Y. and Fuchs, E., Cell Regulation, 1:791-809 (1990)). TGF-.beta.1 messenger RNAs are expressed in terminally differentiating epidermal cells in vivo (Thomson, N. L., Flanders, K. C., Smith, J. M., Ellingsworth, L. R., Roberts, A. B. and Sporn, M. B., J. Cell. Biol., 108:661-669 (1989)). Furthermore, TGF-.beta.2 and the TGF-.beta.-related gene, Vgrl, are expressed at the time that stratification and keratinization in developing mouse epidermis take place (Lyons, R. M., Graycar, J. L., Lee, A., Hashmi, S., Lindquist, P. B. Chen, E. Y., Hogan, B. L. M. and Drynick, R., Proc. Natl. Acad Sci. USA 86: 4554-4558 (1989); Lyons, K. M., Pelton, R. W. and Hogan, B. L., Development 106:759-767 (1988). In spite of this it is not yet clear whether TGF-.beta.s are major regulators of epidermal homeostasis. TGF-.beta.s form part of a large family of molecules and although at first sight it appears that one factor can mimic the action of others, subtle differences often confer specificity and potentiation to different factors in different systems.
A feedback control is involved in epidermal homeostasis. Feedback control of epidermal cell proliferation is suggested by simple evidence. For example, under stress conditions the tissue never disappears although the mitotic activity is considerably depressed. Vice versa, psoriatic epidermis showing an abnormally high proliferative activity does not become thicker and thicker but reaches a homeostatic equilibrium.
This control is not due to contact inhibition. If keratinized or keratinizing cells are removed, for example by means of stripping with adhesive tape (Pinkus, H. J., Invest. Dermatol. 16:383-386 (1951)) or by friction, the underlying basal cells respond by a burst of mitotic activity so that the cell loss is rapidly and locally compensated. It should be noted that even though the basal layer was not altered in this experiment there was a burst of mitotic activity suggesting that contact inhibition is unlikely to be involved in the growth control mechanism of normal epidermis. As in the case of a deeper wound, the enhanced cell proliferation was--apart from a period of overshooting--automatically reduced to its normal value when the repair had been finished.
From this it is obvious that experiments on wound healing and tissue repair can provide insight into the regulatory mechanisms governing epidermal cell proliferation, although the repair of skin lesions is a highly complex process including primarily epithelial cell migration as well as division of epidermal cells and a response of the underlying connective tissue.
In order to provoke the repair process, Bullough and Laurence (Bullough, W. S. and Laurence, E. B. Proc. Roy. Soc. B. 151:517-536 (1960)), made a 1 cm long cut in the dorsal skin of mice which extended down through the panniculus carnosus. This resulted in a stimulation of mitotic activity which reached a peak after 36-48 h. This enhanced proliferative response was almost completely restricted to a zone with a width of 1 mm from the cut edge. Within this zone a gradient of mitotic activity with the highest activity proximal to the wound edge could be observed.
Feedback mitotic control in epidermis appears to be due to the presence of negative growth factors. In a second series of experiments said authors investigated the influence of a small cut made through the epidermis on one side of a mouse ear on the mitotic activity of the epidermis on the opposite side of the ear. A mouse ear is only about 0.15 mm thick and its two epidermal layers are separated by a very thin layer of connective tissue. It could be expected, therefore, that the zone of high mitotic activity adjacent to the wound would extend to the uninjured opposite side of the ear. Indeed, it was observed that the opposite undamaged epidermis showed a mitotic response which was as powerful as that adjacent to the wound. With proper control experiments, this effect was shown not to be due to the greatly enriched blood supply around the damaged area. The authors were thus left with the conclusion that the enhanced mitotic activity in the vicinity of the wound as well as on the opposite side of the ear was the consequence of either the production of a mitogenic agent (wound hormone, (Abercrombie, M., Symposium Soc. Exp. Biol. 11:235 (1957)) or the loss of an endogenous inhibitor which is synthesized within the skin.
Bullough and Laurence (Bullough, W. S. and Laurence, E. B. Proc. Roy. Soc. B. 151:517-536 (1960)) made a wound in the subcutaneous tissue including hair roots without damaging the overlying epidermis. Under these conditions no significant mitotic response of the epidermis was observed (though the wounded area developed a rich blood supply and suffered a heavy invasion of leucocytes). If the wound was not only a simple cut but a more extended lesion, the highest mitotic activity on the undamaged opposite side was found opposite to the center of the lesion. The authors considered this observation not to be consistent with the assumption of a wound hormone thought to be released from the wound edges but took it as strong evidence for the loss of a pre-existing mitotic inhibitor. They thus proposed that normally an inhibitory substance is in constant production, and that it may be lost partly with the cornified cells which are shed from the surface and partly by diffusion into the dermis. It may also be unstable. In the neighborhood of a wound there appears to be both a reduced inhibitor production and a drainage away of inhibitor into the wound (Bullough, W. S. and Laurence, E. B. Proc. Roy. Soc. B. 151:517-536 (1960)). Iversen et al. (Iversen, O. H., Bhangoo, K. S. and Hansen, K., Virch. Arch. B. 16:157-159 (1974)) repeated and extended this type of experiment on wound healing in an impressive study using the web membrane of the African fruit bat. Since this tissue resembles a mouse ear in many aspects (the two epidermal layers are separated by a thin connective tissue sheet with a diameter of 1 mm or less) but is much larger and easier to handle, it was found to be an almost ideal object for the purpose envisaged.
When an area of epidermis was removed from the ventral side of the web by stripping with adhesive tape, several waves of increased mitotic activity and the development of epidermal hyperplasia were observed adjacent to the wound as well as on the central region of the undamaged opposite side. Furthermore, retransplantation of the epidermis to the stripped area (which in fact should have increased the sources of putative wound hormone) prevented development of hyperplasia opposite the wound.
Hyperproliferative epidermal conditions.
There have been many studies attempting to describe and quantify the cell proliferation patterns of normal and diseased skin. Normal epidermis has a very low mitotic activity with cells cycling every 200-300 hours. Yet when the epidermis is wounded a burst of mitotic activity takes place so that the cells divide up to ten times faster depending on the conditions and the severity of the wound (Pinkus, H. J., Invest. Dermatol. 16:383-386 (1951); Bullough, W. S. and Laurence, E. B. Proc. Roy. Soc. B. 151: 517-536 (1960)). In contrast, human hair root cells are rapidly proliferating cells with cell cycle times in the order of 35 hours. Whilst the data on psoriatic epidermis is more controversial there is general agreement that psoriasis is a disease characterized by epidermal cell hyperproliferation and incomplete keratinization (Weinstein, G. D., Colton, A. and McCulloh, J. L., In: Psoriasis Cell Proliferation. Eds. Wright, N., Camplejohn, R. S. Churchill Livingstone (1983)). Estimation of cell cycle times for psoriasis vary from group to group and depend on the methods used. Weinstein et al. have conducted a study of normal and psoriatic epidermis in vivo using the frequency of labeled mitosis method and have reported that whilst the cell cycle time of normal skin is about 300 hours, involved psoriatic epidermal cells have a cell cycle time of about 37 hours (Weinstein, G. D., Colton, A. and McCulloh, J. L., In: Psoriasis Cell Proliferation. Eds. Wright, N., Camplejohn, R. S. Churchill Livingstone (1983)).
The accessibility of cutaneous and genital epithelial tumors has permitted use of the fraction of labeled mitosis (FLM) method to study cell kinetics in these tissues. In basal cell carcinoma and in squamous cell carcinoma kinetic data have been obtained which show kinetic parameters comparable to those of hyperproliferative skin conditions. However, a second peak in the FLM curves (which would have given a direct estimation of the cell cycle times), has not been reported.
Members of the TGF-.beta. superfamily induce differentiation in several tissues.
There is accumulating evidence that TGF-.beta.-related genes are important regulators of many morphogenetic events. TGF-.beta.1, .beta.2 and .beta.3 have been implicated in murine embryogenesis. Another group of the TGF-.beta. family whose members show greatest homology to the drosophila gene DPP and the Xenopus gene Vg1 includes Bone Morphogenetic proteins (BMP) (Thomson, N. L., Flanders, K. C., Smith, J. M., Ellingsworth, L. R., Roberts, A. B., and Sporn, M. B., J. Cell Biol., 108: 661-669 (1989); Lyons, R. M., Graycar, J. L., Lee, A., Hashmi, S., Lindquist, P. B. Chen, E. Y., Hogan, B. L. M. and Drynick, R., Proc. Natl. Acad Sci. USA 86:4554-4558 (1989)) (osteogenin), and 2b (now known as BMP-4) as well as the murine Vgrl, osteogenic protein 1, and GDF-1 (Jones, M. C., Lyons, K. M. and Hogan, B. L. M., Development 111:531542 (1991)). Another subgroup of the TGF-.beta.-related molecules are the activins. The activins were initially found to elicit FSH release (Mason, A. J., Hayflick, J. S., Ling, N., Esch F., Ueno, N., Ying, S. Guillemin, R., Niall, H. and Seeburg, P. H., Nature, 318: 659-663 (1985); Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W. Karr, D. and Spiess, J., Nature 321:776-779 (1986); Ling, N., Ying, S. Y., Ueno, N., Shimasaki, S., Esch F., Hotta, M. and Guillemin, R., Nature 321:779-782 (1986)). Two forms of activins have been isolated and shown to be either disulfide-linked homodimer of the inhibin .beta.A subunit (activin-A) or a heterodimer composed of a .beta.A and a .beta.B subunit (activin AB) (Mason, A. J., Hayflick, J. S., Ling, N., Esch F., Ueno, N., Ying, S. Guillemin, R., Niall, H. and Seeburg, P. H., Nature, 318:659-663 (1985); Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W. Karr, D. and Spiess, J., Nature 321:776-779 (1986); Ling, N., Ying, S. Y., Ueno, N., Shimasaki, S., Esch F., Hotta, M. and Guillemin, R., Nature 321:779-782 (1986)). Activin A has also been found to stimulate erythroid differentiation (Yu, J., Shao, L., Lemas, V., Yu, A. L., Vaughan, J., Rivier, J. and Vale, W. Nature 330:765-767 (1987)) and to promote neuron survival in vivo (Schubert, D., Kimura, H., LaCorbiere, M., Vaughan, J., Karr, D. and Fisher, W. H., Nature 344:868-870 (1990)). Activins, both A and B, have now been shown to induce the formation of mesodermal structures in Xenopus and chicks (Asashima, M., Nakano, H., Shimade, K., Kinoshita, K., Ishii, K., Shibai, H. and Ueno, N., Roux's Arch. Dev. Biol. 198:330-335 (1990); Smith, J. C. M., Price, B. M. J., Van Nimmen, K. and Huylecroeck, D. Nature 345:729-731 (1990); Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J., Vale, W. and Melton, D.C. Cell 63:485-493 (1990); Mitrani, E. and Shimoni, Y., Science 247:1092-1094 (1990); Mitrani, E., Ziv., T., Thomsen, G., Shimoni, Y., Melton D. A., and Bril. A., Cell 63:495-501 (1990)).
Recently, the activins have been found to have strong differentiation capacities on embryonic primary ectodermal cells (Asashima, M., Nakano, H., Shimade, K., Kinoshita, K., Ishii, K., Shibai, H. and Ueno, N., Roux's Arch. Dev. Biol. 198:330-335 (1990); Smith, J. C. M., Price, B. M. J., Van Nimmen, K. and Huylecroeck, D. Nature 345:729-731 (1990)). Activin A can induce axial mesoderm in Xenopus and our group has shown that activin B is probably the endogenous inductor of axial structures in birds (Mitrani, E., Ziv., T., Thomsen, G., Shimoni, Y., Melton D. A., and Bril. A., Cell 63:495-501 (1990)). Also very recently Mathews and Vale (1991) reported the cloning of the activin receptor. This receptor seems to have common features with other growth factor receptors but in contrast to others it is the first receptor to display serine kinase activity (Mathews, L. S., Vale, W. W. Cell 65: 973-982 (1991)).
Tumor Suppressor Genes are involved in the control of cell proliferation.
Tumor formation arises as a consequence of alterations in the control of cell proliferation and disorders in the interactions between cells and their surroundings that result in invasion and metastasis. A breakdown in the relationship between increase in cell number resulting from cell division and withdrawal from the cell cycle due to differentiation or cell death lead to disturbances in the control of cell proliferation. In normal tissues, homeostasis is maintained by ensuring that as each stem cell divides only one of the two daughters remains in the stem cell compartment, while the other is committed to a pathway of differentiation (Cairns, J., Nature 255:197-200 (1975)). The control of cell multiplication will therefore be the consequence of signals affecting these processes. These signals may be either positive or negative, and the acquisition of tumorigenicity results form genetic changes that affect these control points.
It has now been possible to characterize some of these control points and to identify the genetic changes that contribute to malignancy. In the best-studied examples, changes at several different genes have been shown to occur within each tumor. These alterations affect genes concerned with positive stimuli to growth and genes whose products are normally involved in inhibition of cell growth (reviewed in Fearon, E. R. and Vogelstein, B., Cell 61:759-767 (1990)).
Tumors have a defined pattern of growth.
The patterns of growth of a variety of rapidly growing, transplantable and malignant tumors of epithelial origin are not random conglomerations of cells but organized tissues with characteristic histological patterns. The establishment of the basic pattern depends, first, on the connective-tissue-adjacent position of the mitotic cells, and second, on the distal movement, aging and finally death of the differentiated cells. In any tumor these normal rules persist. When growth begins a solid tumor typically consists of an outer sheath of connective-tissue-adjacent mitotic cells, a medial sheath of differentiated aging cells and an inner mass of dead cells. Except that the tumor forms a cyst instead of a sheet, the cells are stratified as they are, for instance, in the epidermis. The picture presented by a typical solid tumor is consistent with a situation in which there is an excessive number of mitotic-cycle cells and an inadequate number of post-mitotic aging cells. Thus, cell production continues to exceed cell loss.
Removal of one tumor accelerates growth of a second identical tumor.
When two identical tumors are present, the removal of one accelerates the growth of the other (Goodman, G. J., Proc. Amer. Assoc. Cancer Res. 2:207- (1957)); partial hepatectomy stimulates the growth of adenomatous hepatic nodules (Trotter, N. L., Cancer Res. 21:778 (1961)). When two tumors of different tissue origins are present together in the same animal, they grow independently of each other so that each reaches its usual plateau as if the other was not present (Burns, E. R., Growth 33:25 (1969)). This last experiment neatly disproves the idea that tumor growth is inhibited by nutrient exhaustion or by toxic metabolic products.
The most extensive studies of the plateau phenomenon in tumors are those of Bichel (Bichel, P., Eur. J. Cancer 6:291 (1970); Bichel, P. Eur. J. Cancer 8:167 (1972); Bichel, P., Nat. Cancer Inst. Mon. 38:197 (1973)), who carefully plotted the growth characteristics of three mouse ascites tumors, each derived from a different tissue and each reaching a stable plateau without killing its host. He found that the removal of tumor cells in the plateau phase causes the immediate resumption of growth of the remaining cell mass; that the cell-free ascites fluid, taken at the plateau phase and injected into another mouse, inhibits mitotic activity in tumor cells in the rapid growth phase, but only if these tumor cells are of the same type of tumor; that when two different tumors are grown simultaneously in the same mouse, each grows at its normal rate to reach its normal plateau irrespective of the presence of the other; and that, when two tumors are grown simultaneously in the same mouse, the cell-free ascites fluid from another mouse containing only one of the tumors inhibits the growth of only the same type of tumor leaving the other tumor to continue its uninhibited growth.
Activin A, and the gene encoding therefor, are known, inter alia, from European Patent Application No. 222,491. This publication discloses the synthesis of Activin A by recombinant techniques and its use in the manipulation of fertility in animals.